RVA Vaccines Research Articles

Epidemiology of group A rotavirus in children under five years of age with gastroenteritis in N'Djamena, Chad.

Group A Rotaviruses (RVA) is one of the most common causes of severe diarrhoea in infants and children under 5 years of age. Unlike many countries in the world where RVA surveillance/control is active, in Chad , there is currently no applied RVA immunization program and surveillance strategy. The present study aims to determine the prevalence and associated risk factors of RVA gastroenteritis among children under five years of age in N'Djamena. This study comprised two parts: (1) A cross-sectional study carried in four hospitals in N'Djamena between August and November 2019, to determine infection risk factors and evidence of RVA infection among children aged five and below, consulted or hospitalized for diarrhea. An ELISA based RVA VP6 protein detection was used to determine RVA infection prevalence. Infection results and sociodemographic data were statistically analysed to determine RVA infection risk factors. (2) A retrospective study that consisted of analysing the records of stool examinations of the period from January 2016 to December 2018, to determine the prevalence of infectious gastroenteritis among the target population. For the cross-sectional study, RVA infection prevalence was 12.76% (18/141) with males (61.11%) being more affected (sex ratio: 1.57). Children below 12 months were the most affected age group (44.44%) and 44.4% were malnourished. The mean Vesikari score shows that 38.8% of children have a high severity level and 41.1% have a moderate level. For the retrospective study, 2,592 cases of gastroenteritis hospitalization were analysed; 980 out of 2,592 cases (37.81%) of hospitalization due to diarrhoea were due to diarrhoeagenic pathogens including Emtamoeba hystolitica, Gardia lamblia, Trichomonas hominis, Hymenolepis nana, Escherichia coli, Shigella spp, Proteus mirabilis, and Klebsiella oxytoca. Cases of diarrhoea with negative pathogen search were 1,612 cases (62.19%). The diarrhoea peak was observed during the dry seasons, and the age group under 11 months was the most affected was (57.3%). This study describes the evidence of RVA infection among diarrhoeic children below five years of age in N'Djamena, thus indicates a serious health burden. Malnourishment younger age was the higher risk factor. Further studies are needed to determine the circulating strains prior to considering introduction of RVA vaccine and setup a routine rotavirus surveillance in Chad.

Emergence of Intergenogroup Reassortant G9P[4] Strains Following Rotavirus Vaccine Introduction in Ghana.

Rotavirus (RVA) is a leading cause of childhood gastroenteritis. RVA vaccines have reduced the global disease burden; however, the emergence of intergenogroup reassortant strains is a growing concern. During surveillance in Ghana, we observed the emergence of G9P[4] RVA strains in the fourth year after RVA vaccine introduction. To investigate whether Ghanaian G9P[4] strains also exhibited the DS-1-like backbone, as seen in reassortant G1/G3/G8/G9 strains found in other countries in recent years, this study determined the whole genome sequences of fifteen G9P[4] and two G2P[4] RVA strains detected during 2015-2016. The results reveal that the Ghanaian G9P[4] strains exhibited a double-reassortant genotype, with G9-VP7 and E6-NSP4 genes on a DS-1-like backbone (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). Although they shared a common ancestor with G9P[4] DS-1-like strains from other countries, further intra-reassortment events were observed among the original G9P[4] and co-circulating strains in Ghana. In the post-vaccine era, there were significant changes in the distribution of RVA genotype constellations, with unique strains emerging, indicating an impact beyond natural cyclical fluctuations. However, reassortant strains may exhibit instability and have a limited duration of appearance. Current vaccines have shown efficacy against DS-1-like strains; however, ongoing surveillance in fully vaccinated children is crucial for addressing concerns about long-term effectiveness.

Characterization and immunogenicity of a novel chimeric hepatitis B core-virus like particles (cVLPs) carrying rotavirus VP8*protein in mice model

Given the efficacy and safety issues of the WHO for approved/prequalified live attenuated rotavirus (RV) vaccines, studies on alternative non-replicating modals and proper RV antigens are actively undertaken. Herein, we report the novel chimeric hepatitis B core-virus like particles (VLPs) carrying RV VP8*26-231 protein of a P [8] strain (cVLPVP8*), as a parenteral VLP RV vaccine candidate. SDS-PAGE and Western blotting analyses indicated the expected size of the E. coli-derived HBc-VP8* protein that self-assembled to cVLPVP8* particles. Immunization in mice indicated development of higher levels of IgG and IgA as well as higher IgG1/IgG2a ratios by cVLPVP8* vaccination compared to the VP8* alone. Assessment of neutralizing antibodies (nAbs) indicated development of heterotypic nAbs with cross-reactivity to a heterotypic RV strain by cVLPVP8* immunization compared to VP8* alone. The observed anti-VP8* cross-reactivity might indicate the possibility of developing a Pan-genomic RVA vaccine based on the cVLPVP8* formulation that deserves further challenge studies.

Surveillance of human Group A rotavirus in Ningxia, China (2015-2021): Emergence and prevalence of G9P[8]-E2 and G3P[8]-E2 genotypes.

Group A rotaviruses (RVA) are the primary pathogens of acute gastroenteritis. Currently, two live attenuated RVA vaccines, LLR and RotaTeq, have been introduced into mainland China but are not included in the national immunization program. Because of the unknown genetic evolution of group A rotavirus in an all-age population in Ningxia, China, we monitored the epidemiological characteristics and circulating genotypes of RVA as a reference for developing vaccine strategies. We conducted seven years of consecutive surveillance of RVA based on stool samples from patients with acute gastroenteritis in sentinel hospitals in Ningxia, China, from 2015 to 2021. Reverse transcription quantitative polymerase chain reaction(RT-qPCR) was used to detect RVA in stool samples. Genotyping and phylogenetic analysis of VP7, VP4 and NSP4 genes were performed by reverse transcription-polymerase chain reaction(RT-PCR) and nucleotide sequence determination. RVA was detected in 16.58% (1436/8662) of 8662 stool samples. The positive rates were 7.17% (201/2805) and 21.09% (1235/5857) in adults and children, respectively. The most affected age group was infants and children aged 12-23months, with a positive rate of 29.53% (p<0.05). A significant winter/spring seasonality was observed. 23.29% positive rate in 2020 was the highest in 7years (p<0.05). The region with the highest positive rate in the adult group was Yinchuan, and the children's group was Guyuan. A total of 9 genotype combinations were found to be distributed in Ningxia. The dominant genotype combinations in this region gradually changed from G9P[8]-E1, G3P[8]-E1, G1P[8]-E1 to G9P[8]-E1, G9P[8]-E2, and G3P[8]-E2 during these seven years. Rare strains (e.g., G9P[4]-E1, G3P[9]-E3 and G1P[8]-E2) were occasionally detected during the study. During the study period, changes in the significant RVA circulating genotype combinations and the emergence of reassortment strains were observed, particularly the emergence and prevalence of G9P[8]-E2, G3P[8]-E2 reassortants in the region. These results indicate the importance of continuous monitoring of the molecular evolution and recombination characteristics of RVA, and should not be limited to G/P genotyping but should consider multi-gene fragment co-analysis and whole genome sequencing.

Predominance of DS‐1‐like G8P[8] rotavirus reassortant strains in children hospitalized with acute gastroenteritis in Thailand, 2018–2020

AbstractRotavirus A (RVA) is an important cause of acute gastroenteritis (AGE) in children. This study aims to investigate the molecular epidemiology of RVA in children hospitalized with AGE in Chiang Rai, Thailand in 2018–2020 by reverse transcription polymerase chain reaction. Of 302 samples, RVA was detected in 11.6% (35 samples): 11.3% (19/168) in 2018–2019 and 11.9% (16/134) in 2019–2020. G8P[8] was the predominant genotype at 68.4% in 2018–2019 and 81.2% in 2019–2020. G1P[8] (15.8%), G2P[4] (5.3%), G3P[8] (10.5%) in 2018–2019, and G9P[8] (18.8%) in 2019–2020 were also detected. Whole‐genome analysis of G8P[8] revealed a DS‐1‐like genetic backbone: G8‐P[8]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2. Phylogenetically, the VP7 genes of G8P[8] clustered in a major lineage with previously published 51 DS‐1‐like G8P[8] reference strains, closely related to 13 G8P[8] strains from Thailand and China. These G8P[8] strains contained two unique amino acid substitutions (A125S and N147D) in the VP7 antigenic epitopes. In addition, the VP1 and NSP2 genes of G8P[8] clustered in lineages separated from the DS‐1‐like G8P[8] reference strains with a high genetic divergence but were closely related to G1P[8], G2P[4], G3P[8], or G9P[8]. Several amino acid differences were observed in the VP7 and VP8* antigenic epitopes of G8P[8] compared with RVA vaccine strains. Homology modeling confirmed that these different amino acid residues were located on the surface‐exposed area of the structure. Taken together, the genetic analysis clearly defines the Chiang Rai DS‐1‐like G8P[8] strains as a novel reassortant strain that might have evolved genetically through reassortment events and consequently received their VP1 and NSP2 genes from locally cocirculating‐RVA genotypes.

Phylogenetic analysis of the viral proteins VP4/VP7 of circulating human rotavirus strains in China from 2016 to 2019 and comparison of their antigenic epitopes with those of vaccine strains.

Group A rotaviruses (RVAs) are the most common etiological agents of severe acute diarrhea among children under 5 years old worldwide. At present, two live-attenuated RVA vaccines, LLR (G10P[15]) and RotaTeq (G1–G4, G6 P[8], P[5]), have been introduced to mainland China. Although RVA vaccines can provide homotypic and partially heterotypic protection against several strains, it is necessary to explore the genetic and antigenic variations between circulating RVAs and vaccine strains. In this study, we sequenced viral protein VP7 and VP4 outer capsid proteins of 50 RVA strains circulating in China from 2016 to 2019. The VP7 and VP4 sequences of almost all strains showed high homology to those of previously reported human strains and vaccine strains of the same genotype. However, in the presumed antigenic epitopes of the VP7 and VP4, multiple amino acid variations were found, regardless of the G and P genotypes of these strains. Moreover, all circulating G3 RVA strains in China potentially possess an extra N-linked glycosylation site compared with the G3 strain of RotaTeq. The potential N-linked glycosylation site at residues 69–71 was found in all G9 strains in China but not in the G9 strain of the Rotavac or Rotasill vaccine. These variations in antigenic sites might result in the selection of strains that escape the RVA neutralizing-antibody pressure imposed by vaccines. Furthermore, the G4 and P[6] genotypes in this study showed high homology to those of porcine strains, indicating the transmission of G4 and P[6] genotypes from pigs to humans in China. More genetic surveillance with antigenic evaluation in prevalent RVAs is necessary for developing and implementing rotavirus vaccines in China.

Differentiation between Wild-Type Group A Rotaviruses and Vaccine Strains in Cases of Suspected Horizontal Transmission and Adverse Events Following Vaccination.

Human group A rotaviruses (RVA) are important enteric pathogens, as they are a leading cause of acute gastroenteritis (AGE) in children worldwide. Since 2013, the German Standing Committee on vaccination recommended the routine rotavirus vaccination for infants in Germany. While vaccination has significantly decreased RVA cases and worldwide mortality, in some cases, infants can develop acute gastroenteritis as an adverse reaction after immunization with an attenuated live vaccine. Pediatricians, as well as clinicians and diagnostic laboratories, contacted the Consultant Laboratory for Rotaviruses and inquired whether cases of RVA-positive AGE after vaccination were associated with vaccine or with wild-type RVA strains. A testing algorithm based on distinguishing PCRs and confirmative sequencing was designed, tested, and applied. Diagnostic samples from 68 vaccinated children and six cases where horizontal transmission was suspected were investigated in this study. Using a combination of real-time PCR, fragment-length analysis of amplicons from multiplex PCRs and confirmative sequencing, vaccine-like virus was detected in 46 samples and wild-type RVA was detected in 6 samples. Three mixed infections of vaccine and wild-type RVA were detectable, no RVA genome was found in 19 samples. High viral loads (>1.0 × 107 copies/g stool) were measured in most RVA-positive samples. Furthermore, information on co-infections with other AGE pathogens in the vaccinated study population was of interest. A commercial multiplex PCR and in-house PCRs revealed three co-infections of vaccinated infants with bacteria (two samples with Clostridioides difficile and one sample with enteropathogenic E. coli) and six co-infections with norovirus in a subset of the samples. Human astrovirus was detected in one sample, with suspected horizontal transmission. The cases of suspected horizontal transmission of vaccine RVA strains could not be confirmed, as they either involved wild-type RVA or were RVA negative. This study shows that RVA-positive AGE after vaccination is not necessarily associated with the vaccine strain and provides a reliable workflow to distinguish RVA vaccine strains from wild-type strains.

Diversity of rotavirus strains circulating in Haiti before and after introduction of monovalent vaccine

BackgroundHaiti introduced a monovalent human group A rotavirus (RVA) vaccine (Rotarix) into its routine infant immunization program in April 2014. The goal of the surveillance program was to characterize RVA strains circulating in Haiti before and after RVA vaccine introduction. MethodsStool samples were collected from children <5 years old presenting with acute gastroenteritis at 16 hospitals in Haiti. RVA antigen enzyme immunoassay (EIA) testing was performed, and G and P genotypes were determined for positive specimens. In this study, genotype data for samples collected from May 2012 through April 2014 (the pre-vaccine introduction era) and May 2014 through July 2019 (post-vaccine introduction era) were analyzed. ResultsA total of 809 specimens were tested by the Centers for Disease Control and Prevention. During the pre-vaccine introduction era (May 2012 through April 2014), G12P[8] was the predominant genotype, detected in 88–94% of specimens. There was a high prevalence of the equine-like G3P[8] genotype among Haitian children with RVA after vaccine introduction. ConclusionsThe predominance of equine-like G3P[8] in three of five RVA seasons post-vaccine introduction suggests possible vaccine-specific selection pressure in Haiti. These temporal variations in RVA genotype predominance will require continued monitoring in Haiti as the vaccination program continues.

Genetic and immunological characterization of G9 group Aporcine rotaviruses in China.

G9 group A rotaviruses (RVAs) are considered emerging pathogens in pigs and humans, and pigs are considered a potential host reservoir for human G9 RVAs. In this study, RVAs of two genotypes, G9P[23] and G9P[13], were successfully isolated and the genomic sequences were obtained, the genome constellation is G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 respectively. One strain which amplified from clinic faecal sample had an unique genome constellation G9-P[23]-I1-R1-C1-M1-A8-N1-T1-E1-H1. All the genomic segments of three porcine G9 RVAs were closely related to those of porcine and/or porcine-like human RVAs, demonstrating that the three viruses were porcine-human reassortant strains. To study the immunogenicity of the porcine G9 RVAs, 6-week-old female BALB/c mice were immunized with inactivated vaccines derived from porcine RVAs and then mated. The highest titres of neutralizing antibodies against G9P[23] and G9P[13] porcine RVAs (1,291 ± 35.22 and 1:232 ± 39.28 respectively) were produced in mice 7 days after the second immunization. Suckling mice born to the vaccinated dams were protected by maternal antibodies against challenge with homologous strains. Overall, our data demonstrate the occurrence of porcine-human reassortants of G9 RVAs, and extend our understanding of the immunogenicity of porcine G9 rotaviruses. They also provide a basis for the development of a porcine G9 RVA vaccine.

Prevalence and genotypes distribution of group A rotavirus among outpatient children under 5 years with acute diarrhea in Shanghai, China, 2012\u20132018

BackgroundGroup A rotavirus (RVA) remains the main causative agent of acute diarrhea among children under five years in countries that have not yet introduced the RVA vaccine worldwide. Long-term and continuous monitoring data on RVA infection in outpatient children were lacking in Shanghai. We investigated the prevalence and distribution of RVA genotypes in outpatient children with acute diarrhea in Shanghai from 2012 to 2018.MethodsStool specimens of outpatient children under five years were collected from the Children’s Hospital of Fudan University in Shanghai, China. All the samples enrolled in this study were detected and characterized for the P and G genotypes of RVA were determined using the semi-multiplex RT-PCR technique.ResultsOf 1814 children enrolled with acute diarrhea and having specimens collected, 246 (13.6%) were infected with RVA. The highest frequency of RVA was observed in children younger than two years old (87.0%, 214/246). Year-round RVA transmission was observed and the RVA detection rate peaked every winter and troughed in summer. In this study, 12 different RVA strains were identified in children. G9P[8] (49.2%, 121/246) was detected as the most prevalent genotype, followed by G–P[8] (22.8%, 56/246), G3P[8] (11.4%, 28/246), and G9P- (4.9%, 12/246). Although RVA strains detected in this study varied with the time, G9P[8] has been the most predominant circulating genotype since 2012. Furthermore, 12.2% (30/246) RVA positive samples were co-infected with other diarrhea viruses.ConclusionThe present analysis showed that RVA was still a major cause of children with acute diarrhea in Shanghai from 2012 to 2018. A great diversity of RVA strains circulated in children with acute diarrhea with G9P[8] being the predominant genotype since 2012. Long-term and continuous monitoring of RVA genotypes is therefore indispensable to refine future vaccine strategy in Shanghai.

Evolutionary changes between pre- and post-vaccine South African group A G2P[4] rotavirus strains, 2003-2017.

The transient upsurge of G2P[4] group A rotavirus (RVA) after Rotarix vaccine introduction in several countries has been a matter of concern. To gain insight into the diversity and evolution of G2P[4] strains in South Africa pre- and post-RVA vaccination introduction, whole-genome sequencing was performed for RVA positive faecal specimens collected between 2003 and 2017 and samples previously sequenced were obtained from GenBank (n=103; 56 pre- and 47 post-vaccine). Pre-vaccine G2 sequences predominantly clustered within sub-lineage IVa-1. In contrast, post-vaccine G2 sequences clustered mainly within sub-lineage IVa-3, whereby a radical amino acid (AA) substitution, S15F, was observed between the two sub-lineages. Pre-vaccine P[4] sequences predominantly segregated within sub-lineage IVa while post-vaccine sequences clustered mostly within sub-lineage IVb, with a radical AA substitution R162G. Both S15F and R162G occurred outside recognised antigenic sites. The AA residue at position 15 is found within the signal sequence domain of Viral Protein 7 (VP7) involved in translocation of VP7 into endoplasmic reticulum during infection process. The 162 AA residue lies within the hemagglutination domain of Viral Protein 4 (VP4) engaged in interaction with sialic acid-containing structure during attachment to the target cell. Free energy change analysis on VP7 indicated accumulation of stable point mutations in both antigenic and non-antigenic regions. The segregation of South African G2P[4] strains into pre- and post-vaccination sub-lineages is likely due to erstwhile hypothesized stepwise lineage/sub-lineage evolution of G2P[4] strains rather than RVA vaccine introduction. Our findings reinforce the need for continuous whole-genome RVA surveillance and investigation of contribution of AA substitutions in understanding the dynamic G2P[4] epidemiology.

Genetic characterization of P[8] rotavirus strains circulated in Iran between 2009 and 2017.

Group A rotavirus (RVA) is the most common cause of acute gastroenteritisworldwide, which is responsible for causing an estimated 120 000 deaths in children under 5 years of age, which mostly occur in the lower-income countries of Asia and Africa. The G1P[8] is a common genotype of RVA that has spread throughout the world, including Iran and this genotype is present in two commonly used RVA vaccines, RotarixTM and RotaTeqTM . In this study, we investigated the genetic diversity, viral evolution, and differences between antigenic epitopes of Iran's P[8] strains and two licensed vaccines. The phylogenetic and evolutionary analysis was carried out, using MEGA version 6.0 and BEAST, respectively. Antigenic epitopes of VP8* were compared to determine the differences between strains from Iran and RotarixTM and RotaTeqTM . The P[8]-lineages III and IV were found as the predominant P genotype that circulated in Iran. The TMRCA of P[8]-lineages III and IV was estimated in 1987 and 2009, respectively. The P[8]-lineage III strains showed 12 amino acid changes compared to RotarixTM and 10 amino acid changes compared to RotaTeqTM . The P[8]-lineage IV strains showed 10 amino acid variations for both RotarixTM and RotaTeqTM strains. The results revealed that the P[8] strains circulating in Iran differ from RotarixTM and RotaTeqTM strains. To monitor the long-term effects of vaccines on the emergence of P[8] strains with different lineages, routine and successful monitoring of these strains will be crucial.

Surveillance and molecular characterization of human sapovirus in patients with acute gastroenteritis in Brazil, 2010 to 2017.

Human sapoviruses (HuSaV) are associated with acute gastroenteritis (AGE), causing sporadic cases and outbreaks in patients worldwide. In Brazil, however, there are few reports describing the prevalence of HuSaV in patients with AGE. Describing the diversity of HuSaV in Brazil by detecting and molecularly characterizing HuSaV among patients with AGE during an 8-year period (2010-2017). A total of 3974 stool samples, testing negative for rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV), were selected and screened for the presence of HuSaV. Nested RT-PCR were performed for a partial region of VP1, sequenced and genetic analyzed for genotyping the positive samples. In the current study, the HuSaV prevalence was determined to be 3.7% (149/3974). A higher prevalence, 5.7% (118/2074), was observed in children under 2 years of age. During the surveillance period, 13 outbreaks were detected: 12 outbreaks in children under 3 years old and one outbreak in adults. Among the 149 HuSaV positive cases, 106 samples (71%) were successfully sequenced. The most prevalent genotype found was GI.1 (44.3%), followed by GI.2 (21.7%), GI.3 (3.8%), GI.6 (2.8%), GII.1 (5.7%), GII.2 (8.5%), GII.3 (2.8%), GII.4 (2.8%), GII.5 (5.7%) and GIV.1 (1.9%). Two GIV.1 strains characterized in this study are, to date, the only strains of this genotype reported in Brazil. The present study elucidated the circulation of HuSaV in Brazil and highlight that HuSaV has not assumed an epidemiological importance in the country after the introduction of the RVA vaccine.

Comparative genomic analysis of genogroup 1 and genogroup 2 rotaviruses circulating in seven US cities, 2014-2016.

For over a decade, the New Vaccine Surveillance Network (NVSN) has conducted active rotavirus (RVA) strain surveillance in the USA. The evolution of RVA in the post-vaccine introduction era and the possible effects of vaccine pressure on contemporary circulating strains in the USA are still under investigation. Here, we report the whole-gene characterization (eleven ORFs) for 157 RVA strains collected at seven NVSN sites during the 2014 through 2016 seasons. The sequenced strains included 52 G1P[8], 47 G12P[8], 18 G9P[8], 24 G2P[4], 5 G3P[6], as well as 7 vaccine strains, a single mixed strain (G9G12P[8]), and 3 less common strains. The majority of the single and mixed strains possessed a Wa-like backbone with consensus genotype constellation of G1/G3/G9/G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while the G2P[4], G3P[6], and G2P[8] strains displayed a DS-1-like genetic backbone with consensus constellation of G2/G3-P[4]/P[6]/P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Two intergenogroup reassortant G1P[8] strains were detected that appear to be progenies of reassortment events between Wa-like G1P[8] and DS-1-like G2P[4] strains. Two Rotarix® vaccine (RV1) and two RV5 derived (vd) reassortant strains were detected. Phylogenetic and similarity matrices analysis revealed 2-11 sub-genotypic allelic clusters among the genes of Wa- and DS-1-like strains. Most study strains clustered into previously defined alleles. Amino acid (AA) substitutions occurring in the neutralization epitopes of the VP7 and VP4 proteins characterized in this study were mostly neutral in nature, suggesting that these RVA proteins were possibly under strong negative or purifying selection in order to maintain competent and actual functionality, but fourteen radical (AA changes that occur between groups) AA substitutions were noted that may allow RVA strains to gain a selective advantage through immune escape. The tracking of RVA strains at the sub-genotypic allele constellation level will enhance our understanding of RVA evolution under vaccine pressure, help identify possible mechanisms of immune escape, and provide valuable information for formulation of future RVA vaccines.

Molecular characterization of human group A rotavirus genotypes circulating in Rawalpindi, Islamabad, Pakistan during 2015-2016.

Group A rotaviruses (RVA) are one of the major causes of acute gastroenteritis (AGE) in young children worldwide. Owing to lack of proper surveillance programs and health facilities, developing countries of Asia and Africa carry a disproportionately heavy share of the RVA disease burden. The aim of this hospital-based study was to investigate the circulation of RVA genotypes in Rawalpindi and Islamabad, Pakistan in 2015 and 2016, prior to the implementation of RVA vaccine. 639 faecal samples collected from children under 10 years of age hospitalized with AGE were tested for RVA antigen by ELISA. Among 171 ELISA positive samples, 143 were successfully screened for RT-PCR and sequencing. The prevalence of RVA was found to be 26.8% with the highest frequency (34.9%) found among children of age group 6–11 months. The most predominant circulating genotypes were G3P[8] (22.4%) followed by G12P[6] (20.3%), G2P[4] (12.6%), G1P[8] (11.9%), G9P[6] (11.9%), G3P[4] (9.1%), G1P[6] (4.2%), G9P[8] (4.2%), and G3P[6] (0.7%). A single mixed genotype G1G3P[8] was also detected. The findings of this study provide baseline data, that will help to assess if future vaccination campaigns using currently available RVA vaccine will reduce RVA disease burden and instigate evolutionary changes in the overall RVA biology. The high prevalence of RVA infections in Pakistan require to improve and strengthen the surveillance and monitoring system for RVA. This will provide useful information for health authorities in planning public health care strategies to mitigate the disease burden caused by RVA.

Molecular Epidemiology and Clinical Features of Rotavirus Infection Among Pediatric Patients in East Java, Indonesia During 2015-2018: Dynamic Changes in Rotavirus Genotypes From Equine-Like G3 to Typical Human G1/G3.

Group A rotavirus (RVA) is the most important cause of severe gastroenteritis among children worldwide, and effective RVA vaccines have been introduced in many countries. Here we performed a molecular epidemiological analysis of RVA infection among pediatric patients in East Java, Indonesia, during 2015–2018. A total of 432 stool samples were collected from hospitalized pediatric patients with acute gastroenteritis. None of the patients in this cohort had been immunized with an RVA vaccine. The overall prevalence of RVA infection was 31.7% (137/432), and RVA infection was significantly more prevalent in the 6- to 11-month age group than in the other age groups (P < 0.05). Multiplex reverse transcription-PCR (RT-PCR) revealed that the most common G-P combination was equine-like G3P[8] (70.8%), followed by equine-like G3P[6] (12.4%), human G1P[8] (8.8%), G3P[6] (1.5%), and G1P[6] (0.7%). Interestingly, the equine-like strains were exclusively detected until May 2017, but in July 2017 they were completely replaced by a typical human genotype (G1 and G3), suggesting that the dynamic changes in RVA genotypes from equine-like G3 to typical human G1/G3 in Indonesia can occur even in the country with low RVA vaccine coverage rate. The mechanism of the dynamic changes in RVA genotypes needs to be explored. Infants and children with RVA-associated gastroenteritis presented more frequently with some dehydration, vomiting, and watery diarrhea, indicating a greater severity of RVA infection compared to those with non-RVA gastroenteritis. In conclusion, a dynamic change was found in the RVA genotype from equine-like G3 to a typical human genotype. Since severe cases of RVA infection were prevalent, especially in children aged 6 to 11 months or more generally in those less than 2 years old, RVA vaccination should be included in Indonesia’s national immunization program.

First Detection of DS-1-like G1P[8] Double-gene Reassortant Rotavirus Strains on The American Continent, Brazil, 2013

Emergence of DS-1-like-G1P[8] rotavirus in Asia have been recently reported. We report for the first time the detection and the whole genome phylogenetic analysis of DS-1-like-G1P[8] strains in America. From 2013 to 2017, a total of 4226 fecal samples were screened for rotavirus by ELISA, PAGE, RT-PCR and sequencing. G1P[8] represented 3.7% (30/800) of all rotavirus-positive samples. DS-1-like-G1P[8] comprised 1.6% (13/800) detected exclusively in 2013, and Wa-like-G1P[8] comprised 2.1% (17/800) detected from 2013 to 2015. Whole genome sequencing confirmed the DS-1-like backbone I2-R2-C2-M2-A2-N2-T2-E2-H2. All genome segments of the Brazilian DS-1-like-G1P[8] strains clustered with those of Asian strains, and apart from African DS-1-like-G1P[8] strains. In addition, Brazilian DS-1-like-G1P[8] reassortants distantly clustered with DS-1-like backbone strains simultaneously circulating in the country, suggesting that the Brazilian DS-1-like-G1P[8] strains are likely imported from Asia. Two distinct NSP4 E2 genotype lineages were also identified, indicating the existence of a co-circulating pool of different DS-1-like G1P[8] strains. Surveillance systems must be developed to examine if RVA vaccines are still effective for the prevention against unusual DS-1-like-G1P[8] strains.

Rotavirus VP6 as a potential vaccine candidate.

By the age of 5years, virtually all children have been infected by group A rotavirus (RVA), which is responsible for around half million mortality annually prior to vaccination. Relatively high rate of the morbidity and mortality highlights the necessity of applying preventive procedures particularly in developing countries. Two live attenuated RVA vaccines (Rotarix and RotaTeq) are licensed and now being used in many countries worldwide. Although these vaccines are shown to reduce the mortality up to 50%, several key questions yet remained to answer. Indeed, the licensed RV vaccines were found to be less effective in countries of sub-Saharan Africa and Southeast Asia. Therefore, developing next generation RVA vaccines is warranted. VP6 is highly abundant and conserved protein that forms the middle layer of RV particles and was shown to be both antigenic and immunogenic. Although it does not induce neutralizing antibodies, different VP6 preparations were found to induce homologous and cross-reactive immune responses with partial protection from RVA replication. Although the molecular mechanisms are not fully elucidated, VP6-based RVA vaccine candidates are worthy of further consideration. This review aims to focus on different aspects of VP6 protein and its potentiality for an alternative RV vaccine against RV disease.

Surveillance and molecular characterization of human adenovirus in patients with acute gastroenteritis in the era of rotavirus vaccine, Brazil, 2012–2017

BackgroundIn Brazil, limited data are available regarding the incidence and molecular epidemiology of Human Adenovirus (HAdV) in diarrheic disease, especially in the post rotavirus (RVA) vaccine era. ObjectiveThe aims of the study were to investigate the frequency of HAdV infections in patients with gastroenteritis during a 6-year period (2012–2017); conduct molecular typing of positive strains, and obtain further information on the HAdV seasonality. Study designA total of 3003 fecal samples negative for both, RVA and Norovirus, were selected and tested for HAdV by PCR. Positive HAdV samples were sequenced to obtain genotype identification. ResultsHAdV was detected in 3.9% (117/3003); 76 belong to species F (70 HAdV-F41; 6 HAdVF40), 31 to species C (15 HAdV-C1; 13 HAdV-C2; 3 HAdV-C5), 5 belong to species D (3 HAdVD56 and 2 untyped), 4 belong to species A (2 HAdV-A12; 2 HAdV-A31), and 1 belong to HAdVB3. Detection rate significantly varied according to the year, suggesting that HAdV infections show a tendency to occur in natural oscillatory fluctuation. No consistent seasonal pattern was identified. Children ≤5 years exhibited higher positivity rate, reinforcing that HAdV is an important pathogen in childhood diarrhea. Genetic analysis indicated that HAdV strains circulating in Brazil were closely related to worldwide strains, and there is no evidence for the introduction of a particular HAdV variant in the country. ConclusionsThe present investigation does not suggest that HAdV has assumed an epidemiological importance in Brazil after the RVA vaccine introduction and contributed to the definition of the clinical and public health significance of HAdV infections.

Spread of the emerging equine-like G3P[8] DS-1-like genetic backbone rotavirus strain in Brazil

Background: In 2013, a novel equine-like G3P[8] DS-1 like rotavirus (RVA) emerged as dominant strain worldwide. In 2016, this strain was reported in Brazil. The aims of the present study were to conduct a genetic retrospective investigation since 2013 in order to identify the possible entry of this novel G3P[8] strain in Brazil, describe its distribution across the country, help the understanding of evolutionary dynamics and potential implications in RVA vaccine programs. Methods & Materials: Between January/2013 and October/2017, a total of 4025 fecal specimens were collected from Southern, Southeastern and Midwest regions, and likely to be representative of Brazilian population. Specimens were screened for RVA using ELISA, and genotyped by RT-PCR. VP7 amplicons of positive G3P[8] samples were submitted to sequencing in order to identify the equine-like G3P[8] strains by nucleotide similarity search. Six equine-like G3P[8] representative samples were selected for investigation of the whole genome. Results: G3P[8] represented 16.7% (123/736) of all RVA-positive samples, and further divided as equine-like G3 (6.4%; 47/736) and wild-type G3 (11.5%; 85/736). During 2013-2014, wild-type G3P[8] was the dominant strain across Brazil, and no equine-like G3P[8] was detected. Equine-like G3P[8] strain was first identified in March/2015 in Paraná State, suggesting that the strain stepped into Brazil through Southern region. The equine-like G3P[8] circulated only in Paraná till February/2016, but rapidly spread across the country after that, reaching Amazonas in March, Goiás in July and the Federal District in September. Equine-like G3P[8] strain reached São Paulo in August/2017. No wild-type G3 was detected since March/2015, therefore, it could be speculated that the atypical G3P[8] genotype was able to completely replace wild-type G3P[8] strains. Whole genome analysis revealed a DS-1 like genotype constellation (G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and segments shared the highest nucleotide sequence identity with strains isolated in Brazil and Spain. Conclusion: This study highlights the emergence of the atypical equine-like G3P[8] genotype circulating in Brazil, and reinforces the potential for novel human/animal reassortant to arise within human population. Equine-like G3P[8] DS-1-like strains pose a challenge for the RVA surveillance, once G/P typing characterization by RT-PCR is unable to identify this intergenogroup reassortant without inclusion of sequencing analysis.